Cholesterol metabolism is associated with soluble amyloid precursor protein production in Alzheimer's disease

Disturbances of the cholesterol metabolism are associated with Alzheimer's disease (AD) risk and related cerebral pathology. Experimental studies found changing levels of cholesterol and its metabolites 24S‐hydroxycholesterol (24S‐OHC) and 27‐hydroxycholesterol (27‐OHC) to contribute to amyloidogenesis by increasing the production of soluble amyloid precursor protein (sAPP). The aim of this study was to evaluate the relationship between the CSF and circulating cholesterol 24S‐OHC and 27‐OHC, and the sAPP production as measured by CSF concentrations of sAPP forms in humans. The plasma and the CSF concentrations of cholesterol, 24S‐OHC and 27‐OHC, and the CSF concentrations of sAPPα, sAPPβ, and Aß1‐42 were assessed in subjects with AD and controls with normal cognition. In multivariate regression tests including age, gender, albumin ratio, and apolipoprotein E (APOE)ε4 status CSF cholesterol, 24S‐OHC, and 27‐OHC independently predicted the concentrations of sAPPα and sAPPβ. The associations remained significant when analyses were separately performed in the AD group. Furthermore, plasma 27‐OHC concentrations were associated with the CSF sAPP levels. The results suggest that high CSF concentrations of cholesterol, 24S‐OHC, and 27‐OHC are associated with increased production of both sAPP forms in AD.     

The role of presenilin-1 in the gamma-secretase cleavage of the amyloid precursor protein of Alzheimer's disease

Presenilin-1 (PS1) is required for the release of the intracellular domain of Notch from the plasma membrane as well as for the cleavage of the amyloid precursor protein (APP) at the gamma-secretase cleavage site. It remains to be demonstrated whether PS1 acts by facilitating the activity of the protease concerned or is the protease itself. PS1 could have a gamma-secretase activity by itself or could traffic APP and Notch to the appropriate cellular compartment for processing. Human APP 695 and PS1 were coexpressed in Sf9 insect cells, in which endogenous gamma-secretase activity is not detected. In baculovirus-infected Sf9 cells, PS1 undergoes endoproteolysis and interacts with APP. However, PS1 does not cleave APP in Sf9 cells. In CHO cells, endocytosis of APP is required for Abeta secretion. Deletion of the cytoplasmic sequence of APP (APPDeltaC) inhibits both APP endocytosis and Abeta production. When APPDeltaC and PS1 are coexpressed in CHO cells, Abeta is secreted without endocytosis of APP. Taken together, these results conclusively show that, although PS1 does not cleave APP in Sf9 cells, PS1 allows the secretion of Abeta without endocytosis of APP by CHO cells.     

Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes

Accumulation of extracellular amyloid beta peptide (Abeta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Abeta from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in Abeta production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to Abeta. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to Abeta.     

Pin1 promotes production of Alzheimer's amyloid beta from beta-cleaved amyloid precursor protein

Here we show that prolyl isomerase Pin1 is involved in the Abeta production central to the pathogenesis of Alzheimer's disease. Enzyme immunoassay of brains of the Pin1-deficient mice revealed that production of Abeta40 and Abeta42 was lower than that of the wild-type mice, indicating that Pin1 promotes Abeta production in the brain. GST-Pin1 pull-down and immunoprecipitation assay revealed that Pin1 binds phosphorylated Thr668-Pro of C99. In the Pin1-/- MEF transfected with C99, Pin1 co-transfection enhanced the levels of Abeta40 and Abeta42 compared to that without Pin1 co-transfection. In COS7 cells transfected with C99, Pin1 co-transfection enhanced the generation of Abeta40 and Abeta42, and reduced the expression level of C99, facilitating the C99 turnover. Thus, Pin1 interacts with C99 and promotes its gamma-cleavage, generating Abeta40 and Abeta42. Further, GSK3 inhibitor lithium blocked Pin1 binding to C99 by decreasing Thr668 phosphorylation and attenuated Abeta generation, explaining the inhibitory effect of lithium on Abeta generation.     

Proteolytic processing of the Alzheimer's disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases

Alzheimer's disease is characterized by neurodegeneration and deposition of betaA4, a peptide that is proteolytically released from the amyloid precursor protein (APP). Missense mutations in the genes coding for APP and for the polytopic membrane proteins presenilin (PS) 1 and PS2 have been linked to familial forms of early-onset Alzheimer's disease. Overexpression of presenilins, especially that of PS2, induces increased susceptibility for apoptosis that is even more pronounced in cells expressing presenilin mutants. Additionally, presenilins themselves are targets for activated caspases in apoptotic cells. When we analyzed APP in COS-7 cells overexpressing PS2, we observed proteolytic processing close to the APP carboxyl terminus. Proteolytic conversion was increased in the presence of PS2-I, which encodes one of the known PS2 pathogenic mutations. The same proteolytic processing occurred in cells treated with chemical inducers of apoptosis, suggesting a participation of activated caspases in the carboxyl-terminal truncation of APP. This was confirmed by showing that specific caspase inhibitors blocked the apoptotic conversion of APP. Sequence analysis of the APP cytosolic domain revealed a consensus motif for group III caspases ((IVL)ExD). Mutation of the corresponding Asp664 residue abolished cleavage, thereby identifying APP as a target molecule for caspase-like proteases in the pathways of programmed cellular death.     

The amyloid precursor protein of Alzheimer's disease is released by human platelets

Western blots of normal human platelets, employing a monoclonal antibody raised against the full-length amyloid precursor protein of Alzheimer's disease (APP695), revealed major bands of 100-110 and 120-130 kDa in both cytosolic, membrane, and released fractions. These species were similar in size to forms seen in brain preparations and in plasma. There was no difference in Western blots of platelet preparations from Alzheimer patients compared with controls. Purified platelet amyloid precursor proteins were sequenced and shown to be amino terminally homogeneous. Immunohistochemistry localized the antigen to the platelet and megakaryocyte and demonstrated weak immunostaining of some lymphocytes. Immunoprecipitation of material released from platelets demonstrated that sedimentable full-length APP with the carboxyl-terminal epitope, and soluble APP lacking the carboxyl-terminal epitope, may exist in the circulation. Western blots and carboxyl-terminal and amino-terminal APP radioimmunoassay of material released by platelets in response to stimulation revealed that platelets release APP during degranulation. The function of platelet APP is yet to be determined, but the present studies suggest a role in regulation of the coagulation cascade or in platelet aggregation.     

Copper promotes the trafficking of the amyloid precursor protein

Accumulation of the amyloid β peptide in the cortical and hippocampal regions of the brain is a major pathological feature of Alzheimer disease. Amyloid β peptide is generated from the sequential protease cleavage of the amyloid precursor protein (APP). We reported previously that copper increases the level of APP at the cell surface. Here we report that copper, but not iron or zinc, promotes APP trafficking in cultured polarized epithelial cells and neuronal cells. In SH-SY5Y neuronal cells and primary cortical neurons, copper promoted a redistribution of APP from a perinuclear localization to a wider distribution, including neurites. Importantly, a change in APP localization was not attributed to an up-regulation of APP protein synthesis. Using live cell imaging and endocytosis assays, we found that copper promotes an increase in cell surface APP by increasing its exocytosis and reducing its endocytosis, respectively. This study identifies a novel mechanism by which copper regulates the localization and presumably the function of APP, which is of major significance for understanding the role of APP in copper homeostasis and the role of copper in Alzheimer disease.     

Plasmalogen synthesis is regulated via alkyl-dihydroxyacetonephosphate-synthase by amyloid precursor protein processing and is affected in Alzheimer's disease

Lipids play an important role as risk or protective factors in Alzheimer's disease, which is characterized by amyloid plaques composed of aggregated amyloid-beta. Plasmalogens are major brain lipids and controversially discussed to be altered in Alzheimer's disease (AD) and whether changes in plasmalogens are cause or consequence of AD pathology. Here, we reveal a new physiological function of the amyloid precursor protein (APP) in plasmalogen metabolism. The APP intracellular domain was found in vivo and in vitro to increase the expression of the alkyl-dihydroxyacetonephosphate-synthase (AGPS), a rate limiting enzyme in plasmalogen synthesis. Alterations in APP dependent changes of AGPS expression result in reduced protein and plasmalogen levels. Under the pathological situation of AD, increased amyloid-beta level lead to increased reactive oxidative species production, reduced AGPS protein and plasmalogen level. Accordingly, phosphatidylethanol plasmalogen was decreased in the frontal cortex of AD compared to age matched controls. Our findings elucidate that plasmalogens are decreased as a consequence of AD and regulated by APP processing under physiological conditions.     

Berberine alters the processing of Alzheimer's amyloid precursor protein to decrease Abeta secretion

Berberine is an isoquinoline alkaloid isolated from Coptidis rhizoma, a major herb widely used in Chinese herbal medicine. Berberine's biological activity includes antidiarrheal, antimicrobial, and anti-inflammatory effects. Recent findings show that berberine prevents neuronal damage due to ischemia or oxidative stress and that it might act as a novel cholesterol-lowering compound. The accumulation of amyloid-beta peptide (Abeta) derived from amyloid precursor protein (APP) is a triggering event leading to the pathological cascade of Alzheimer's disease (AD); therefore the inhibition of Abeta production should be a rational therapeutic strategy in the prevention and treatment of AD. Here, we report that berberine reduces Abeta levels by modulating APP processing in human neuroglioma H4 cells stably expressing Swedish-type of APP at the range of berberine concentration without cellular toxicity. Our results indicate that berberine would be a promising candidate for the treatment of AD.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

Modeling of substrate specificity of the Alzheimer's disease amyloid precursor protein beta-secretase

The enzyme BACE (beta-site APP-cleaving enzyme) has recently been identified as the beta-secretase that cleaves the amyloid precursor protein (APP) to produce the N terminus of the Abeta peptide found in plaques in the brains of Alzheimer's disease patients. BACE is an aspartic protease similar to pepsin and renin. Comparative modeling of the three-dimensional structure of BACE in complex with its substrate shows that several residues confer specificity of the enzyme for APP. In particular, Arg296 forms a salt-bridge with the P1' Asp of the APP substrate, explaining the unusual preference of BACE among aspartic proteases for a P1' residue that is negatively charged. Several hydrophobic residues in the enzyme form a pocket for the P1 hydrophobic residue (Met in wild-type APP and Leu in APP with the "Swedish mutation" associated with early-onset of Alzheimer's disease). Inhibitors that can bind to the BACE active site may prove useful for drugs to treat and prevent Alzheimer's disease.     

Protein kinase C and amyloid precursor protein processing in skin fibroblasts from sporadic and familial Alzheimer's disease cases

Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimer's disease cases. In contrast, familial Alzheimer's disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.     

Protease-specificity of Kunitz inhibitor domain of Alzheimer's disease amyloid protein precursor

The putative inhibitor domain of Alzheimer's disease amyloid protein precursor was purified from E. coli containing a synthetic gene encoding the Kunitz domain. The purified protein (A4 inhibitor) inhibited the activity of trypsin, forming a 1:1 molar complex with the enzyme. It also strongly inhibited plasmin (Ki = 7.5 x 10(-11) M) from human serum and tryptase (Ki = 2.2 x 10(-10) M) from rat mast cells (tryptase M). In addition, it inhibited rat pancreatic trypsin, alpha-chymotrypsin and kallikrein and human serum kallikrein, but did not inhibit rat chymase, pancreatic elastase, alpha-thrombin, urokinase, papain or cathepsin B.     

Multiple effects of aspartate mutant presenilin 1 on the processing and trafficking of amyloid precursor protein

PS1 deficiency and expression of PS1 with substitutions of two conserved transmembrane aspartate residues ("PS1 aspartate variants") leads to the reduction of Abeta peptide secretion and the accumulation of amyloid precursor protein (APP) C-terminal fragments. To define the nature of the "dominant negative" effect of the PS1 aspartate variants, we stably expressed PS1 harboring aspartate to alanine substitutions at codons 257 (D257A) or 385 (D385A), singly or in combination (D257A/D385A), in mouse neuroblastoma, N2a cells. Expression of the PS1 aspartate variants resulted in marked accumulation of intracellular and cell surface APP C-terminal fragments. While expression of the D385A PS1 variant reduced the levels of secreted Abeta peptides, we now show that neither the PS1 D257A nor D257A/D385A variants impair Abeta production. Surprisingly, the stability of both immature and mature forms of APP is dramatically elevated in cells expressing PS1 aspartate variants, commensurate with an increase in the cell surface levels of APP. These findings lead us to conclude that the stability and trafficking of APP can be profoundly modulated by coexpression of PS1 with mutations at aspartate 257 and aspartate 385.     

Aging impact on amyloid precursor protein neuronal trafficking

Neurons live a lifetime. Neuronal aging may increase the risk of Alzheimer's disease. How does neuronal membrane trafficking maintain synapse function during aging? In the normal aged brain, intraneuronal beta-amyloid (Aβ) accumulates without Alzheimer's disease mutations or risk variants. However, do changes with neuronal aging potentiate Aβ accumulation? We reviewed the membrane trafficking of the amyloid precursor protein in neurons and highlighted its importance in Aβ production. Importantly, we reviewed the evidence supporting the impact of aging on neuronal membrane trafficking, APP processing, and consequently Aβ production. Dissecting the molecular regulators of APP trafficking during neuronal aging is required to identify strategies to delay synaptic decline and protect from Alzheimer's disease.